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DNA kits:

 DNA 1000DNA 7500DNA 12000
Sizing range, bp25-1000100-7500100-12000
Quantitative range0.1-50 ng/µl0.5-50 ng/µl0.5-50 ng/µl

RNA kits:

 RNA Nano 6000RNA Pico 6000
Quantitative range25-500 ng/μl — total RNA assay
25-250 ng/μl — mRNA assay
Qualitative range5-500 ng/μl — total RNA assay
25-250 ng/μl — mRNA assay
50-5000 pg/μl (in water) — total RNA
250-5000 pg/μl (in water) — mRNA

Reagents and equipment:

  • RNaseZAP® for electrode decontamination (Ambion, Inc. cat. no. 9780)
  • Four electrode cleaners (two for RNase — free water and two for RNaseZAP)
  • RNase-free water
  • Pipettes (10 μl and 1000 μl) with compatible tips
  • 0.5 ml LoBind tubes (RNase-free)
  • Microcentrifuge (≥ 1300 g)
  • Heating block or water bath for ladder/sample preparation

[править] Setting up the Chip Priming Station

  1. Adjust the base plate
    For all DNA and RNA kits the base plate of chip priming station should be in position C.
  2. Adjust the syringe clip

[править] Essential Measurement Practices

  • Allow all reagents and samples to equilibrate to room temperature for 30 minutes before use.
  • Protect dye and dye mixtures from light. Remove light covers only when pipetting. The dye decomposes when exposed to light and this reduces the signal intensity.
  • Always insert the pipette tip to the bottom of the well when dispensing the liquid. Placing the pipette at the edge of the well may lead to poor results.
  • Use loaded chips within 5 minutes after preparation. Reagents might evaporate, leading to poor results.

[править] Cleaning the Electrodes before Running Assays

Repeat twice:

  • Fill one electrode cleaner with 350µl of RNaseZAP.
  • Open the lid and place the electrode cleaner in the Agilent 2100 bioanalyzer.
  • Close the lid and leave it closed for 1 minutes.
  • Open the lid and remove the electrode cleaner. Label the electrode cleaner and keep it for future use.
  • Fill another electrode cleaner with 350 μl of fresh RNase-free water.
  • Open the lid and place the electrode cleaner in the Agilent 2100 bioanalyzer.
  • Close the lid and leave it closed for 1 minutes.

Wait another 30 seconds to allow the water on the electrodes to evaporate before closing the lid.

[править] Sample preparation

For accurate determination of DNA concentration, the total amound of DNA in sample must be between 0.5-50 ng/μl. If concentration is excessively high, dilute to 0.5-50 ng/μl.

[править] Preparing the Gel-Dye Mix

  • Allow DNA dye concentrate (blue cap) and DNA gel matrix (red cap) to equilibrate to room temperature for 30 min.
  • Vortex DNA dye concentrate (blue cap) and add 25 μl of the dye to a DNA gel matrix vial (red cap).
  • Vortex solution well and spin down. Transfer to spin filter.
  • Centrifuge at 1500 g ± 20% for 10 min. Protect solution from light. Store at 4°C.

[править] Loading the Gel-Dye Mix

  • Allow the gel-dye mix equilibrate to room temperature for 30 min before use.
  • Put a new DNA chip on the chip priming station.
  • Pipette 9.0 μl of gel-dye mix in the well marked File:BioAnalyzer(2).png.
  • Make sure that the plunger is positioned at 1 ml and then close the chip priming station.
  • Press plunger until it is held by the clip.
  • Wait for exactly 30 s then release clip.
  • Wait for 5 s. Slowly pull back plunger to 1ml position.
  • Open the chip priming station and pipette 9.0 μl of gel-dye mix in the wells marked File:BioAnalyzer, (4).png.

[править] Loading the Markers

  • Pipette 5 μl of marker (green cap) in all 12 sample and ladder wells. Do not leave any wells empty.

[править] Loading the Ladder and the Samples

  • Pipette 1 μl of DNA ladder (yellow cap) in the well marked File:BioAnalyzer(5).png .
  • In each of the 12 sample wells pipette 1 μl of sample (used wells) or 1 μl of de-ionized water (unused wells).
  • Put the chip horizontally in the adapter and vortex for 1 min at the indicated setting (2400rpm).
  • Run the chip in the Agilent 2100 bioanalyzer within 5 min.

[править] Starting the Chip Run

  • In the Instrument context, select the appropriate assay from the Assay menu.
  • Click the Start button in the upper right of the window to start the chip run. The incoming raw signals are displayed in the Instrument context.
  • To enter sample information like sample names and comments, select the Data File link that is highlighted in blue or go to the Assay context and select the Chip Summary tab. Complete the sample name table.
  • To review the raw signal trace, return to the Instrument context

[править] Cleaning Electrodes after a Chip Run

See Cleaning the Electrodes before Running Assays

[править] Preparing the RNA Ladder after Arrival

After reagent kit arrival, spin ladder down. The ladder can be ordered separately (reorder number 5067-1535).
Transfer the ledder in 0.2ml PCR tube.
Heat denature the ladder for 2 min at 70°C in thermal cycler.
Immediately the on ice.
Add 90 μl of RNase-free water and mix thoroughly.
Prepare aliquots in recommended RNase-free vials with the required amount for a typical daily use.
Store aliquots at −70°C.
Before use, thaw ladder aliquots and keep them on ice (avoid extensive warming upon thawing process).

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